THYROID HORMONE RECEPTOR AND GENE EXPRESSION

甲状腺激素受体和基因表达

• 批准号: 2141147
• 负责人: HOWARD C TOWLE
• 金额: $ 11.77万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2141147
• 关键词: DNA binding protein; chemical binding; conformation; dimer; fungal genetics; gel mobility shift assay; gene mutation; genetic regulatory element; genetic transcription; hormone analog; hormone receptor; hormone regulation /control mechanism; molecular cloning; mutant; protein sequence; protein structure function; reporter genes; retinoid binding proteins; thyroid hormones; tissue /cell culture; transcription factor; triiodothyronine

The long range goal of this research is to understand the molecular basis by which the nuclear thyroid hormone receptor (TR) mediates changes in specific gene transcription. TR binds to specific DNA sequences (thyroid hormone response elements or TREs) in both the absence and presence of hormone. Upon binding of hormone, TR undergoes a conformational change that converts it to a transcriptionally active state. The region of TR between the DNA-binding domain and the C-terminal end (residues 170 to 456 of the beta form) is responsible for ligand binding, dimerization and transcriptional activation. Little is currently known regarding these important processes. The focus of this proposal is to identify the critical amino acid residues responsible for these functions and to understand the mechanism of ligand-mediated transcriptional activation by TR. To accomplish these goals, we have established a yeast system in which expression of the reporter gene, beta-galactosidase, is under control of TR. Like many other mammalian transcription factors, TR can function in yeast to activate transcription provided an appropriate DNA recognition site is present. Using the yeast expression system, we have selected for mutations of the C-terminal region of TR that are defective in promoting transcriptional activation. These mutations fall into two general categories: those that fail to bind hormone and those that bind hormone normally, but cannot trans-activate. To evaluate the nature of these functional domains, we will further characterize the mutations leading to defects in hormone-binding and trans-activation. To identify amino acid residues that are in close contact with hormone, we will screen for mutations that alter the ligand-binding specificity of TR (Specific Aim #1). Mutations that bind hormone, but are defective in trans-activation, will be studied to determine whether the defects result from the failure of TR to undergo the ligand-mediated conformational change or lie directly within the trans-activation domain itself (Specific Aim #2). Recently, several laboratories have shown that TR binding and transcriptional activation on certain TREs can be enhanced by heterodimerization with the retinoid X receptor (RXR). We will use the mutations that we have isolated to study the roles of each of the receptor partners in the heterodimer in the activation process (Specific Aim #3). Finally, we will use phenotypic screening in yeast to identify regions of TR that are critical to the process of heterodimerization and to determine whether distinct dimerization domains are involved in interacting with different types of TREs (Specific Aim #4). These studies will allow a better understanding of the process of ligand-mediated transcriptional activation by TR and provide valuable tools for future work to further explore this process.

这项研究的长期目标是了解 甲状腺激素核受体（TR）通过其介导 特异性基因转录TR与特定的DNA序列（甲状腺）结合 激素应答元件或TRE）在不存在和存在下的表达。 激素.当与激素结合时，TR经历构象变化 将其转化为转录活性状态。TR地区 在DNA结合结构域和C末端之间（残基170至456 β形式）负责配体结合、二聚化和 转录激活目前对这些问题知之甚少。 重要进程。本提案的重点是确定 负责这些功能的关键氨基酸残基， 了解配体介导的转录激活机制， tr.为了实现这些目标，我们建立了一个酵母系统， 报告基因β-半乳糖苷酶的表达受 tr.与许多其他哺乳动物转录因子一样，TR可以在哺乳动物中发挥作用。 酵母激活转录提供了适当的DNA识别 现场有。利用酵母表达系统，我们选择了 TR的C-末端区域的突变，其在促进 转录激活这些突变分为两个一般的 类别：那些不能结合激素和那些结合激素 正常但不能反式激活为了评估这些问题的性质， 功能域，我们将进一步表征突变导致 酶结合和反式激活的缺陷。鉴定氨基酸 与激素密切接触的残留物，我们将筛选 改变TR配体结合特异性的突变（特定目的 #1）。结合激素但反式激活有缺陷的突变， 将进行研究，以确定缺陷是否由故障引起 的TR进行配体介导的构象变化或直接位于 在反式激活结构域本身内（具体目标#2）。最近， 几个实验室已经表明，TR结合和转录 在某些TREs上的活化可以通过与异源二聚化来增强。 类维生素A X受体（RXR）。我们将利用我们分离出来的突变体 研究异二聚体中每个受体伴侣的作用， 具体目标#3（Specific Aim #3）最后，我们将使用表型 在酵母中筛选以鉴定TR的区域， 异二聚化的过程，并确定是否不同 二聚化结构域参与与不同类型的 TREs（具体目标#4）。这些研究将有助于更好地了解 配体介导的TR转录激活过程， 为今后进一步探索这一进程提供了宝贵的工具。